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With the application of modern investigative technologies, cholestatic liver diseases of genetic etiology are increasingly identified as the root cause of previously designated "idiopathic" adult and pediatric liver diseases. Here, we review advances in the field enhanced by a deeper understanding of the phenotypes associated with specific gene defects that lead to cholestatic liver diseases. There are evolving areas for clinicians in the current era specifically regarding the role for biopsy and opportunities for a "sequencing first" approach. Risk stratification based on the severity of the genetic defect holds promise to guide the decision to pursue primary liver transplantation versus medical therapy or nontransplant surgery, as well as early screening for HCC. In the present era, the expanding toolbox of recently approved therapies for hepatologists has real potential to help many of our patients with genetic causes of cholestasis. In addition, there are promising agents under study in the pipeline. Relevant to the current era, there are still gaps in knowledge of causation and pathogenesis and lack of fully accepted biomarkers of disease progression and pruritus. We discuss strategies to overcome the challenges of genotype-phenotype correlation and draw attention to the extrahepatic manifestations of these diseases. Finally, with attention to identifying causes and treatments of genetic cholestatic disorders, we anticipate a vibrant future of this dynamic field which builds upon current and future therapies, real-world evaluations of individual and combined therapeutics, and the potential incorporation of effective gene editing and gene additive technologies.

OBJECTIVE Extracorporeal membrane oxygenation (ECMO)-associated direct hyperbilirubinemia (DHB) is likely multifactorial. The objective of this study is to assess the frequency and risk factors for developing direct hyperbilirubinemia while on ECMO, and its implication on the mortality of children. METHODS We performed a retrospective study between January 2010 and January 2020. Using Mayo Clinic electronic health record, we identified children (≤18 years) who required veno-arterial (VA) ECMO support. Demographics, ECMO indication, laboratory findings, and outcomes were abstracted. Illness acuity scores, including vasoactive-ionotropic score (VIS), were used to assess disease severity at time of admission. Study cohort was divided into two groups: children who developed direct hyperbilirubinemia (DHB) on ECMO and children who did not (control). DHB was defined as direct bilirubin (DB) of > 1.0 milligrams/deciliter (mg/dL). Disease acuity and mortality rates were compared between the two groups. Logistic regression was used to analyze the risk of mortality independent of potential confounding variables. RESULTS We identified 106 children who required ECMO support during the study period. Of those, 36 (34%) children developed DHB on ECMO. Illness acuity scores were significantly higher in the DHB group on ECMO day 2 (p = 0.046) and day 7 (p = 0.01). Mortality rate was higher in the DHB group 72%, vs 29% in control group (p

Pediatric acute liver failure (PALF) is a rare, rapidly progressive clinical syndrome with significant morbidity and mortality. The phenotype of PALF manifests as abrupt onset liver dysfunction, which can be brought via disparate etiology. Management is reliant upon intensive clinical care and support, often provided by the collaborative efforts of hepatologists, critical care specialists, and liver transplant surgeons. The construction of an age-based diagnostic approach, the identification of a potential underlying cause, and the prompt implementation of appropriate therapy can be lifesaving; however, the dynamic and rapidly progressive nature of PALF also demands that diagnostic inquiries be paired with monitoring strategies for the recognition and treatment of common complications of PALF. Although liver transplantation can provide a potential life-saving therapeutic option, the ability to confidently determine the certainness that liver transplant is needed for an individual child has been hampered by a lack of adequately tested clinical decision support tools and accurate predictive models. Given the accelerated progress in understanding PALF, we will provide clinical guidance to pediatric gastroenterologists and other pediatric providers caring for children with PALF by presenting the most recent advances in diagnosis, management, pathophysiology, and associated outcomes.

Liver sinusoidal endothelial cells (LSECs) are distinct subtypes of endothelial cells lining a low flow vascular bed at the interface of the liver parenchyma and the circulating immune cells and soluble factors. Emerging literature implicates LSEC in the pathogenesis and progression of nonalcoholic fatty liver disease (NAFLD). During the evolution of NAFLD, LSEC dysfunction ensues. LSECs undergo morphological and functional transformation known as “capillarization,” as well as a pathogenic increase in surface adhesion molecules expression, referred to in this review as “endotheliopathy.” LSECs govern the composition of hepatic immune cell populations in nonalcoholic steatohepatis (NASH) by mediating leukocyte subset adhesion through specific combinations of activated adhesion molecules and secreted chemokines. Moreover, extracellular vesicles released by hepatocyte under lipotoxic stress in NASH act as a catalyst for the inflammatory response and promote immune cell chemotaxis and adhesion. In the current review, we highlight leukocyte adhesion to LSEC as an initiating event in the sterile inflammatory response in NASH. We discuss preclinical studies targeting immune cells adhesion in NASH mouse models and potential therapeutic anti-inflammatory strategies for human NASH.

BACKGROUND AND AIMS Reliance on exception points to prioritize children for liver transplantation (LT) stems from concerns that the Pediatric End-Stage Liver Disease (PELD) score underestimates mortality. Renal dysfunction and serum sodium disturbances are negative prognosticators in adult LT candidates and various pediatric populations, but are not accounted for in PELD. We retrospectively evaluated the effect of these parameters in predicting 90-day wait-list death/deterioration among pediatric patients (

Background & Aims Autophagy plays a crucial role in hepatic homeostasis and its deregulation has been associated with chronic liver disease. However, the effect of autophagy on the release of fibrogenic extracellular vesicles (EVs) by platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) remains unknown. Herein, we aimed to elucidate the role of autophagy, specifically relating to fibrogenic EV release, in fibrosis. Methods In vitro experiments were conducted in primary human and murine HSCs as well as LX2 cells. Small EVs were purified by differential ultracentrifugation. Carbon tetrachloride (CCl4) or bile duct ligation (BDL) were used to induce fibrosis in our mouse model. Liver lysates from patients with cirrhosis or healthy controls were compared by RNA sequencing. Results In vitro, PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) inhibited autophagy and increased HSC-derived EV release. We used this PDGF/SHP2 model to further investigate how autophagy affects fibrogenic EV release. RNA sequencing identified an mTOR (mammalian target of rapamycin) signaling molecule that was regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of multivesicular body-derived exosomes (by inhibiting autophagy) and microvesicles (by activating ROCK1 signaling). These mTOR-dependent EVs promoted in vitro HSC migration. To assess the importance of this mechanism in vivo, SHP2 was selectively deleted in HSCs, which attenuated CCl4- or BDL-induced liver fibrosis. Furthermore, in the CCl4 model, mice receiving circulating EVs derived from mice with HSC-specific Shp2 deletion had less fibrosis than mice receiving EVs from control mice. Correspondingly, SHP2 was upregulated in patients with liver cirrhosis. Conclusion These results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs. Lay summary During liver fibrosis and cirrhosis, activated hepatic stellate cells (HSCs) are the key cell type responsible for fibrotic tissue deposition. Recently, we demonstrated that activated HSCs release nano-sized vesicles enriched with fibrogenic proteins. In the current study, we unveil the mechanism by which these fibrogenic vesicles are released, moving a step closer to the long-term goal of therapeutically targeting this process.

Nonalcoholic hepatitis (NASH) is the progressive inflammatory form of nonalcoholic fatty liver disease. Although the mechanisms of hepatic inflammation in NASH remain incompletely understood, emerging literature implicates the proinflammatory environment created by toxic lipid-induced hepatocyte injury, termed lipotoxicity. Interestingly, numerous NASH-promoting kinases in hepatocytes, immune cells, and adipocytes are activated by the lipotoxic insult associated with obesity. In the current review, we discuss recent advances in NASH-promoting kinases as disease mediators and therapeutic targets. The focus of the review is mainly on the mitogen-activated protein kinases including mixed lineage kinase 3, apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 MAPK; the endoplasmic reticulum (ER) stress kinases protein kinase RNA-like ER kinase and inositol-requiring protein-1α; as well as the Rho-associated protein kinase 1. We also discuss various pharmacological agents targeting these stress kinases in NASH that are under different phases of development.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs’ communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo.Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin β1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease.Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

BACKGROUND In December 2018, United Network for Organ Sharing approved an allocation scheme based on recipients' geographic distance from a deceased donor (acuity circles [ACs]). Previous analyses suggested that ACs would reduce waitlist mortality overall, but their impact on pediatric subgroups was not considered. METHODS We applied Scientific Registry of Transplant Recipients data from 2011 to 2016 toward the Liver Simulated Allocation Model to compare outcomes by age and illness severity for the United Network for Organ Sharing-approved AC and the existing donor service area-/region-based allocation schemes. Means from each allocation scheme were compared using matched-pairs t tests. RESULTS During a 3-year period, AC allocation is projected to decrease waitlist deaths in infants (39 versus 55; P < 0.001), children (32 versus 50; P < 0.001), and teenagers (15 versus 25; P < 0.001). AC allocation would increase the number of transplants in infants (707 versus 560; P < 0.001), children (677 versus 547; P < 0.001), and teenagers (404 versus 248; P < 0.001). AC allocation led to decreased median pediatric end-stage liver disease/model for end-stage liver disease at transplant for infants (29 versus 30; P = 0.01), children (26 versus 29; P < 0.001), and teenagers (26 versus 31; P < 0.001). Additionally, AC allocation would lead to fewer transplants in status 1B in children (97 versus 103; P = 0.006) but not infants or teenagers. With AC allocation, 77% of pediatric donor organs would be allocated to pediatric candidates, compared to only 46% in donor service area-/region-based allocation (P < 0.001). CONCLUSIONS AC allocation will likely address disparities for pediatric liver transplant candidates and recipients by increasing transplants and decreasing waitlist mortality. It is more consistent with federally mandated requirements for organ allocation.

With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity‐inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow‐Chow, pChow‐FFC, pFFC‐Chow, and pFFC‐FFC. Mice were sacrificed at 10 weeks of age. We examined the whole‐liver transcriptome by RNA sequencing (RNA‐seq) and whole‐liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC‐FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC‐FFC mice versus the pChow‐FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC‐Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion: Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

A subset of patients with non-alcoholic fatty liver disease develop an inflammatory condition, termed non-alcoholic steatohepatitis (NASH). NASH is characterised by hepatocellular injury, innate immune cell-mediated inflammation and progressive liver fibrosis. The mechanisms whereby hepatic inflammation occurs in NASH remain incompletely understood, but appear to be linked to the proinflammatory microenvironment created by toxic lipid-induced hepatocyte injury, termed lipotoxicity. In this review, we discuss the signalling pathways induced by sublethal hepatocyte lipid overload that contribute to the pathogenesis of NASH. Furthermore, we will review the role of proinflammatory, proangiogenic and profibrotic hepatocyte-derived extracellular vesicles as disease biomarkers and pathogenic mediators during lipotoxicity. We also review the potential therapeutic strategies to block the feed-forward loop between sublethal hepatocyte injury and liver inflammation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate receptor(s) are up-regulated in human and murine nonalcoholic steatohepatitis (NASH); however, the consequence of this enhanced expression on NASH pathogenesis remains unclear. TRAIL may either accentuate liver injury by promoting hepatic steatosis and inflammation or it may mitigate the disease process by improving systemic insulin resistance and averting hepatic fibrosis. Herein, we investigated the role of TRAIL in an obesity-induced murine model of NASH. C57BL/6 wild-type mice and Trail–/– mice were placed on a 20-week standard chow or a high-fat, high-fructose, and high-cholesterol (FFC) diet, which induces obesity, insulin resistance, and NASH. Metabolic phenotype, liver injury, inflammation and fibrosis, and adipose tissue homeostasis were examined. FFC diet-fed Trail–/– mice displayed no difference in weight gain and metabolic profile when compared to wild-type mice on the same diet. All FFC-fed mice developed significant hepatic steatosis, which was attenuated in Trail–/– mice. TRAIL deficiency also significantly decreased FFC diet-induced liver injury as manifested by reduced serum alanine aminotransferase values, hepatic terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells, and macrophage-associated inflammation. FFC diet-associated hepatic stellate cell activation and hepatic collagen deposition were also abrogated in Trail–/– mice. In contrast to the liver, TRAIL deletion did not improve FFC diet-induced adipose tissue injury and inflammation and actually aggravated insulin resistance. Conclusion: NASH pathogenesis may be dissociated from other features of the metabolic syndrome, and liver-targeted inhibition of TRAIL signaling may be salutary. (Hepatology Communications 2017)

Saturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C-X-C motif chemokine 10 (CXCL10), and release CXCL10-enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3-dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC-treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249-3259, 2017. © 2017 Wiley Periodicals, Inc.

Background & Aims Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin β1 (ITGβ1), which promotes monocyte adhesion and liver inflammation in murine NASH. Methods Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGβ1 neutralizing antibody (ITGβ1Ab) or control IgG isotype. Results Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGβ1. Furthermore, we showed that LPC treatment in hepatocytes activates ITGβ1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGβ1Ab. FFC-fed, ITGβ1Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGβ1Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGβ1Ab treatment significantly ameliorated liver injury and fibrosis. Conclusions Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGβ1-dependent mechanism. ITGβ1Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGβ1 is a potential anti-inflammatory therapeutic strategy in human NASH. Lay summary Herein, we report that a cell adhesion molecule termed integrin β1 (ITGβ1) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGβ1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGβ1 reduces liver inflammation, injury and fibrosis. Hence, ITGβ1 inhibition may serve as a new therapeutic strategy for NASH.

Extracellular vesicles (EVs) are nanometer-sized, membrane-bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell-to-cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano-shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (Hepatology 2016;64:2219-2233).

Background & Aims Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. Methods Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. Results Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow–derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. Conclusions Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.

Relapsing remitting multiple sclerosis (RRMS) is the most prevalent MS subtype. Years after disease onset, most of RRMS patients show transition into secondary progressive form (SPMS). Currently, no biomarkers are available for tracking disease progression. Here, we observed marked elevation of Rho-associated protein kinase 2 (ROCK2) along with significant downregulation of miRNAs 300 and 450b-5p expressions in the serum of 39 RRMS and 35 SPMS Egyptian patients compared to healthy controls. More pronounced alterations were found in SPMS versus RRMS patients. Our findings also suggest relations between elevated ROCK2 and reduced expression of both miRNAs with the degree of disability and disease progression. Notably, these biomarkers effectively discriminated RRMS from SPMS patients with miR-450b-5p showing the highest prognostic power.

Background & Aims Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. Methods C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4μ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. Results Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. Conclusions In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and its cognate receptor(s) are up‐regulated in human and murine nonalcoholic steatohepatitis (NASH); however, the consequence of this enhanced expression on NASH pathogenesis remains unclear. TRAIL may either accentuate liver injury by promoting hepatic steatosis and inflammation or it may mitigate the disease process by improving systemic insulin resistance and averting hepatic fibrosis. Herein, we investigated the role of TRAIL in an obesity‐induced murine model of NASH. C57BL/6 wild‐type mice and Trail –/– mice were placed on a 20‐week standard chow or a high‐fat, high‐fructose, and high‐cholesterol (FFC) diet, which induces obesity, insulin resistance, and NASH. Metabolic phenotype, liver injury, inflammation and fibrosis, and adipose tissue homeostasis were examined. FFC diet‐fed Trail –/– mice displayed no difference in weight gain and metabolic profile when compared to wild‐type mice on the same diet. All FFC‐fed mice developed significant hepatic steatosis, which was attenuated in Trail –/– mice. TRAIL deficiency also significantly decreased FFC diet‐induced liver injury as manifested by reduced serum alanine aminotransferase values, hepatic terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling‐positive cells, and macrophage‐associated inflammation. FFC diet‐associated hepatic stellate cell activation and hepatic collagen deposition were also abrogated in Trail –/– mice. In contrast to the liver, TRAIL deletion did not improve FFC diet‐induced adipose tissue injury and inflammation and actually aggravated insulin resistance. Conclusion: NASH pathogenesis may be dissociated from other features of the metabolic syndrome, and liver‐targeted inhibition of TRAIL signaling may be salutary. (Hepatology Communications 2017;1:648–662)

With the increase in obesity worldwide, its associated comorbidities, including nonalcoholic steatohepatitis (NASH), have become a public health problem that still lacks effective therapy. We have previously reported that mixed-lineage kinase 3-deficient (MLK3-deficient) mice are protected against diet-induced NASH. Given the critical need to identify new therapeutic agents, we sought to examine whether the small-molecule MLK3 inhibitor URMC099 would be effective in reversing diet-induced murine NASH. C57BL/6J mice were fed either a diet high in saturated fat, fructose, and cholesterol (FFC), or a chow diet for 24 weeks. Mice were treated with either URMC099 (10 mg/kg) twice daily by intraperitoneal injection or its vehicle during the last 2 weeks of the feeding study. FFC-fed mice receiving URMC099 had similar body weight, caloric intake, homeostatic model assessment of insulin resistance, metabolic phenotype, and hepatic steatosis compared with vehicle-treated mice. Furthermore, FFC-fed mice treated with URMC099 had less hepatic macrophage infiltration, activation, and proinflammatory polarization, as well as less liver injury and fibrosis when compared with vehicle-treated mice. In conclusion, URMC099 is well tolerated in mice without obvious toxicities and appears to be efficacious in reversing diet-induced NASH. Hence, URMC099 may serve as a therapeutic agent in human NASH.

Hepatocyte injury caused by accumulation of excess lipid intermediates, known as hepatocyte lipotoxicity, is intricately linked to the pathogenesis of obesity-associated nonalcoholic steatohepatitis (NASH). Although both hepatocyte cell injury and inflammation occur in this disease process, the cellular and molecular mechanisms linking hepatocyte injury to inflammation remain unclear. Although multiple cell types contribute to hepatic inflammation in NASH, the predominant cell types appear to be macrophages. In this review, we discuss lipid-induced apoptosis and sublethal signaling events described in lipid-loaded hepatocytes, and how they promote macrophage-associated inflammation. The saturated free fatty acid palmitate and its derivative lysophosphatidylcholine activate cellular apoptotic pathways by upregulating the proapoptotic proteins, including BH3-only protein PUMA and the death receptor TRAIL-R2, which cooperatively promote lipoapoptosis. Hepatocyte apoptosis may contribute to hepatic inflammation and fibrosis via generation of apoptotic bodies. These apoptotic bodies are engulfed by macrophages and stellate cells promoting liver inflammation and fibrosis, respectively. However, recent studies also suggest that proapoptotic, but nonlethal, signaling in hepatocytes leads to the release of extracellular vesicles, which promote hepatic inflammation by inducing macrophage chemotaxis and activation. The vesicle cargo promoting these macrophage processes includes CXCL10, TRAIL, and sphingosine 1-phosphate. Thus, we discuss the possible relative contributions of both hepatocyte apoptotic cell death and nonlethal proapoptotic signaling in generating macrophage-driven hepatic inflammation during NASH.

Pylephlebitis with associated liver abscesses is a rare complication of Crohn's disease that usually presents in the setting of active disease and recent escalation of the immunosuppressive regimen. Here we describe a 17-year-old female patient with active Crohn's disease who presented with a clinical picture and hepatic radiographic findings concerning for pylephlebitis and associated liver abscesses but subsequently confirmed to be an atypical form of focal fatty liver infiltration. We describe the diagnostic difficulty this unique distribution of hepatic fatty infiltration can present and highlight the importance of the different imaging studies to differentiate between atypical focal fatty infiltration and pylephlebitis.

Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, whereas short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short circuiting the normal enterohepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the middistal jejunum for 4–5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis, and endoplasmic reticulum (ER) stress were measured. Rats post-BD lost significantly more weight than the SH rats. BD rats gained less fat mass after surgery. BD rats had improved glucose tolerance, increased higher postprandial glucagon-like peptide-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (C/EBP homologous protein mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/ursodeoxycholic acid [UDCA]) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (C/EBP homologous protein mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase-deficient (Fah(-/-)) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah(-/-) pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH(+) cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah(-/-) liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah(-/-) pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.

Nonalcoholic steatohepatitis (NASH) is an inflammatory lipotoxic disorder, but how inflammatory cells are recruited and activated within the liver is still unclear. We previously reported that lipotoxic hepatocytes release CXCL10-enriched extracellular vesicles, which are potently chemotactic for cells of the innate immune system. In the present study, we sought to determine the innate immune cell involved in the inflammatory response in murine NASH and the extent to which inhibition of the chemotactic ligand CXCL10 and its cognate receptor CXCR3 could attenuate liver inflammation, injury and fibrosis. C57BL/6J CXCL10−/−, CXCR3−/− and wild type (WT) mice were fed chow or high saturated fat, fructose, and cholesterol (FFC) diet. FFC-fed CXCL10−/− and WT mice displayed similar weight gain, metabolic profile, insulin resistance, and hepatic steatosis. In contrast, compared to the WT mice, FFC-fed CXCL10−/− mice had significantly attenuated liver inflammation, injury and fibrosis. Genetic deletion of CXCL10 reduced FFC-induced proinflammatory hepatic macrophage infiltration, while natural killer cells, natural killer T cells, neutrophils and dendritic cells hepatic infiltration were not significantly affected. Our results suggest that CXCL10−/− mice are protected against diet-induced NASH, in an obesity-independent manner. Macrophage-associated inflammation appears to be the key player in the CXCL10-mediated sterile inflammatory response in murine NASH.

Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage-associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone-marrow–derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10-neutralizing antisera. MLK3-deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild-type mice. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages. (Hepatology 2016;63:731–744)

With the obesity epidemic, nonalcoholic fatty liver disease (NAFLD) has become a public health problem with increasing prevalence. The mechanism of disease progression remains obscure and effective therapy is lacking. Therefore, there is a need to understand the pathogenic mechanisms responsible for disease development and progression in order to develop innovative therapies. To accomplish this goal, experimental animal models that recapitulate the human disease are necessary, especially, since causative mechanistic studies of NAFLD are more difficult or unethical to perform in humans. A large number of studies regarding the pathophysiology and treatment of nonalcoholic steatohepatitis (NASH) have been undertaken in mice to model human NAFLD and NASH. This review discusses the known dietary, genetic, and inflammation-based animal models of NASH described in recent years, with a focus on the major advances made in this field.

Backgrounds & Aims Saturated free fatty acids induce hepatocyte lipoapoptosis, a key pathologic feature of non-alcoholic steatohepatitis. The saturated free fatty acid palmitate induces hepatocyte lipoapoptosis via an endoplasmic reticulum stress pathway resulting in c-Jun-N-terminal (JNK) activation. Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase which may also promote JNK activation. Thus, our aim was to determine if GSK-3 inhibition suppresses palmitate induced JNK activation and lipoapoptosis. Methods For these studies, we employed mouse primary hepatocytes, Huh-7 and Hep3B cell lines. Results Palmitate-induced GSK-3 activation was identified by phosphorylation of its substrate glycogen synthase. GSK-3 pharmacologic inhibition, by GSK-3 inhibitor IX and enzastaurin, significantly reduced PA-mediated lipoapoptosis. More importantly, Huh-7 cells, in which either GSK-3α or GSK-3β isoforms were stably and selectively knocked down by shRNA, displayed resistance to palmitate-induced cytotoxicity. GSK-3 pharmacological inhibitors and shRNA-targeted knockdown of GSK-3α or GSK-3β also suppressed JNK activation by palmitate. JNK activation, in part, promotes lipoapotosis by inducing expression of the pro-apoptotic effector p53-upregulated modulator of apoptosis (PUMA). Consistent with this concept, GSK-3 pharmacologic inhibition also reduced PUMA cellular protein levels during exposure to palmitate. On the other hand, the GSK-3 inhibitors did not prevent PA induction of ER stress. Conclusions Our results suggest that GSK-3 activation promotes a JNK-dependent cytotoxic signaling cascade culminating in lipoapoptosis.